Project Summary/Abstract Section Neonatal brain injury is an important cause of death and disability, with pathways of oxidant stress, inflammation, and excitotoxicity leading to damage that progress over a long period of time. Therapies have classically targeted individual pathways during early phases of injury, but targeting pathways later in the injury response may be additionally effective. Therapeutic hypothermia (TH), while being standard of care for hypoxic-ischemic encephalopathy (HIE), provides protection only in 60% of babies. The overarching hypothesis is that the metabolic state of the brain immediately after TH differs markedly between hypothermia responders and non-responders. We will identify the metabolic state after TH using proton and hyperpolarized carbon 13 spectroscopy and then study cellular pathways to identify more precise and individualized treatment approaches. We will use postnatal day 9 mice and follow them through injury evolution. The studies outlined in this proposal reflect an innovative and systematic approach to the study of HI brain injury in the newborn because they combine advanced metabolic imaging techniques (proton and carbon spectroscopy) and cell-signaling studies, focusing on HIF signaling, that will both inform and be informed by human clinical studies. Utilizing genetic cellular approaches in which components of the injury response are specifically deleted/disrupted in the specific cell compartments will allow us to evaluate the benefits of the neurovascular niche in vivo. We will broadly interrogate HIF-dependent signaling pathways following injury using ChIP-Seq. Together, these genetic tools will allow us to explore the molecular regulation of HI both in vitro and in vivo to better identify more appropriate molecular targets for therapy for the individual needing them the most. By investigating responses to HI at a cellular level using traditional biochemical assays and global level in the brain using MR spectroscopy, we aim to make a link between specific cellular changes and metabolic changes that can be detected non-invasively. This approach would eventually allow the findings to be translated into the clinic and potentially change the management of patients. We will use in vitro techniques such as CRISPr/Cas9 and Chip-seq to dissect important signaling pathways like hypoxia inducible factor (HIF). We will also use an invitro approach when we identify appropriate targets and design therapies to counteract deficient repair. Thus, defining the cerebral metabolic signature of non- responders will identify subsequent novel pathways to target, and will lead to improved outcomes that could never be achieved by only targeting pathways through hypothermia alone. Understanding how the cascade of injury responses occur and the key modulators during each phase will lead to more rationale therapies.